title: The separation of flavonoids from Pongamia pinnata using combination columns in high-speed counter-current chromatography with a three-phase solvent system.
abstract: The mangrove plant Pongamia pinnata (Leguminosae) is well known as a plant pesticide. Previous studies have indicated that the flavonoids are responsible of the biological activities of the plant. A new high-speed counter-current chromatography (HSCCC) method for the separation of three flavonoids, karanjin (1), pinnatin (2), and pongaflavone (3), from P. pinnata was developed in the present study. The lower and intermediate phase (LP and IP) of a new three-phase solvent system, n-hexane-acetonitrile-dichloromethane-water, at a volume ratio of 5:5:1:5, were used as the stationary phases, while the upper phase (UP) was used as the mobile phase, and the volume ratio between the stationary phases in the CCC column could be tuned by varying the initial pumped volume ratio of the stationary phases. The CCC columns containing all three phases of the solvent system were considered combination columns. According to the theories of combination column, it is possible to optimize the retention time of the target compounds by varying the volume ratio of the stationary phases in the HSCCC combination columns, as well as the suitable volume ratios of the stationary phases for the separation of the target compounds were predicted from the partition coefficients of the compounds in the three-phase solvent system. Then, three HSCCC separations using the combination columns with initial pumped LP:IP volume ratios of 1:0, 0.9:0.1, and 0.7:0.3 were performed separately based on the prediction. Three target compounds were prepared with high purity when the initial pumped volume ratio of the stationary phases was 0.9:0.1. The baseline separation of compounds 2 and 3 was achieved on the combination column with an initial pumped volume ratio of 0.7:0.3. Furthermore, the three experiments clearly demonstrated that the retentions and resolutions of the target compounds increased with an increasing volume ratio of IP, which is consistent with the prediction for the retention times for the solutes on combination columns. The method proposed here reduces the need for solvent selection compared with the conventional method and may have broad potential applicability in the preparation of natural products.
authors.affiliation: Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, China; Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. Electronic address: yinhao@scsio.ac.cn.
authors.forename: Hao
authors.initials: H
authors.lastname: Yin
authors.forename: Si
authors.initials: S
authors.lastname: Zhang
authors.forename: Lijuan
authors.initials: L
authors.lastname: Long
authors.forename: Hang
authors.initials: H
authors.lastname: Yin
authors.forename: Xinpeng
authors.initials: X
authors.lastname: Tian
authors.forename: Xiongming
authors.initials: X
authors.lastname: Luo
authors.forename: Haihan
authors.initials: H
authors.lastname: Nan
authors.forename: Sha
authors.initials: S
authors.lastname: He
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003377:Countercurrent Distribution / Q000379:methods*; D005419:Flavonoids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D006586:Hexanes / Q000737:chemistry; D008752:Methylene Chloride / Q000737:chemistry; D033641:Millettia / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry; D012997:Solvents / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D005419:Flavonoids; D006586:Hexanes; D010936:Plant Extracts; D012997:Solvents; C026385:n-hexane; D008752:Methylene Chloride; C032159:acetonitrile
keywords: Combination column; High-speed counter-current chromatography (HSCCC); Pongamia pinnata; Three-phase solvent system